Scientists still wonder if animal-derived microbes are prevalent in the environments of people who handle animals, especially when these microbes are suspected of being involved in human disease. The aim of this study was to test a local source tracking database by choosing an environment frequently used by veterinary students and veterinarians and by identifying resident microbiota composition and their sources of contamination (animals, environment, human beings…). The 16S rDNA amplicon sequencing was used to determine the bacterial taxonomic profiles of restroom surfaces. Bacterial sources were identified by linking our metadata-enriched local database to the microbiota profiling analysis using high-quality sequences. Microbiota profiling shows the dominance of four phyla: Actinobacteria, Bacteroidetes, Proteobacteria, and Firmicutes. If the restroom cleaning process did not appear to impact microbiota composition, significant differences regarding bacterial distribution were observed between men and women users in different sampling campaigns. Combining 16S rDNA profiling to our specific sources labeling pipeline we found aquatic and human origins sources were the primary environment keywords in our campaigns. The probable presence of known animal sources (bovids, insects, equids, suids…) associated with bacterial genera such as Chryseobacterium, Bergeyella, Fibrobacter, and Syntrophococcus was also involved in restroom surfaces, emphasizing the proximity between these restrooms and the exchange of bacteria between people involved in animals handling. To summarize, we have demonstrated that DNA sequence-based source tracking may be integrated with high-throughput bacterial community analysis to enrich microbial investigation of potential bacterial contamination sources especially for little known or poorly identified taxa. However, more research is needed to determine the tool's utility in other applications.